The Evolution of Lipidomics during Oil Accumulation of Plukenetia volubilis Seeds

Sacha inchi (Plukenetia volubilis) is a valuable oilseed crop with a high content of polyunsaturated fatty acids (PUFAs). However, there is a lack of in-depth understanding of the lipidomics in Sacha inchi seeds (SIDs). Saturated fatty acids occupied more than half of the proportion (59.31%) in early development, while PUFAs accounted for 78.92% at maturation. The main triacylglycerols were TAG(18:3/18:3/18:3), TAG(18:2/18:2/18:3), and TAG(16:0/18:2/18:2). The corresponding species (18:3/18:3, 18:2/18:2, and 16:0/18:2) were also the main ingredients in diacylglycerol and phosphatidic acid, indicating high PUFA composition in the sn-1 and sn-2 positions of TAG. Only LPC(18:3), LPC(18:2), and LPC(16:0) were identified in SIDs, implying that those PUFAs on the sn-2 positions of the PC(18:3/-), PC(18:2/-), and PC(16:0/-) categories were released into the acyl-CoA pool for the Kennedy pathway. Conversely, the PC(18:1/-) and PC(18:0/-) categories might be responsible for the generation of PC-derived DAG and TAG. The lipidomics data will contribute to understanding the TAG assembly in developing SIDs, especially for PUFAs.


Introduction
Sacha inchi (Plukenetia volubilis), belonging to the family Euphorbiaceae, is a woody liana and is cultivated as an economic crop [1].The oleaginous seeds of this species are the main edible organ for the indigenous people of the Amazon rainforest.Historically, Sacha inchi seeds (SIDs) can be consumed for diverse culinary uses, such as inchi cucho, lechona api, and inchi capi [2].Compositional analysis of mature SIDs indicates that the first main ingredient is oil (33~58%), followed by proteins (22~30%) and other bioactive ingredients [2].It is worth taking note that SID oil was awarded the gold medal at the 'World Edible Oil' contest over three consecutive years from 2004 to 2006 [1].The main fatty acids (FAs) of SID oil were polyunsaturated FAs (PUFAs), including α-linolenic acid (C18:3) and linoleic acid (C18:2) accounting for ∼50% and ∼35% of the total oil content, respectively [3].As the market demand for PUFAs increases, Sacha inchi has become a promising economic crop to widely apply in the production of foods, medications, cosmetics, and other fields [1,4].The development of high-PUFA crops or oils can lead to new market opportunities and economic growth in agriculture and related industries.
In plants, the pathway of oil accumulation is mainly composed of FA biosynthesis, FA desaturation, triacylglycerol (TAG) assembly, and oil body formation [5].In cell plastid, the generated acetyl-CoA is used as a precursor for FA biosynthesis to produce palmitic acid (C16:0) and stearic acid (C18:0).Subsequently, the monounsaturated FAs (MUFAs) and PUFAs are formed in the plastid and endoplasmic reticulum (ER) by corresponding FA desaturases (FADs), respectively [6].In sharp contrast to the clear and single channel of FA biosynthesis and desaturation, TAG assembly is complex and varied.For example, free FAs can be assembled into TAG by the classic Kennedy pathway, the FA chain in phosphatidyl choline (PC) can be interchanged with the FA chain in diacylglycerol (DAG), or the FA chains in PC can also be used for TAG generation [5].The resulting TAG is eventually turned into an oil body for long-term preservation in the seed [7].Although FA compositions are clear in developing SIDs, very little is currently known about lipid changes during oil accumulation.The clarification of lipid composition may lead to an understanding of the processes of PUFA biosynthesis and assembly.
Since the concept of lipidomics was proposed in 2003, lipometabolism has attracted great attention from researchers owing to the innovation of mass spectrometry [8].In this process, lipids are converted into ionized substances measurable by mass spectrometry, thereby enabling the identification of lipid species and their structural characteristics for a comprehensive analysis of all lipid types, their contents, and interrelations within the sample [9].Recent lipidomics in higher plants were applied in the characterization of changes in lipid profiles [9][10][11][12].Lipidomics information during SID oil accumulation is unavailable.The purpose of this study was to investigate the changes in lipid profiles in developing SIDs.This study provides a basis for improving our understanding of FA assembly, in particular PUFA fractions.These findings are informative for breeding programs to enhance PUFA content in crops, thereby improving the nutritional quality of food products.

Oil Content and FA Compositions
Oilseed crops with high yield and oil content are more attractive with increasing demand for vegetable oils.Previous investigations on P. volubilis have revealed a sigmoid pattern of oil accumulation throughout seed development [13,14].Importantly, the pivotal stages of oil accumulation, namely initiation, rapid accumulation, and culmination, occurred at 15, 70, and 110 days after flowering (DAFs), respectively.According to the BBCH scale [15], the three crucial time points of fruit development corresponded to stages 731, 787, and 799, which we selected to explore the lipid compositions (Figure 1a).Our results showed that the SID oil contents displayed a gradual increase with maturity from 2.51% to 55.04% (Figure 1b).This was in line with a previous report that the oil content of mature SIDs ranges from 35 to 60% [3].The fluctuation may be correlated with climatic conditions, cultivation methods, extraction methods, and cultivars [2,16,17].corresponding FA desaturases (FADs), respectively [6].In sharp contrast to the clear and single channel of FA biosynthesis and desaturation, TAG assembly is complex and varied.For example, free FAs can be assembled into TAG by the classic Kennedy pathway, the FA chain in phosphatidyl choline (PC) can be interchanged with the FA chain in diacylglycerol (DAG), or the FA chains in PC can also be used for TAG generation [5].The resulting TAG is eventually turned into an oil body for long-term preservation in the seed [7].Although FA compositions are clear in developing SIDs, very little is currently known about lipid changes during oil accumulation.The clarification of lipid composition may lead to an understanding of the processes of PUFA biosynthesis and assembly.Since the concept of lipidomics was proposed in 2003, lipometabolism has attracted great attention from researchers owing to the innovation of mass spectrometry [8].In this process, lipids are converted into ionized substances measurable by mass spectrometry, thereby enabling the identification of lipid species and their structural characteristics for a comprehensive analysis of all lipid types, their contents, and interrelations within the sample [9].Recent lipidomics in higher plants were applied in the characterization of changes in lipid profiles [9,10,11,12].Lipidomics information during SID oil accumulation is unavailable.The purpose of this study was to investigate the changes in lipid profiles in developing SIDs.This study provides a basis for improving our understanding of FA assembly, in particular PUFA fractions.These findings are informative for breeding programs to enhance PUFA content in crops, thereby improving the nutritional quality of food products.

Oil Content and FA Compositions
Oilseed crops with high yield and oil content are more attractive with increasing demand for vegetable oils.Previous investigations on P. volubilis have revealed a sigmoid pattern of oil accumulation throughout seed development [13,14].Importantly, the pivotal stages of oil accumulation, namely initiation, rapid accumulation, and culmination, occurred at 15, 70, and 110 days after flowering (DAFs), respectively.According to the BBCH scale [15], the three crucial time points of fruit development corresponded to stages 731, 787, and 799, which we selected to explore the lipid compositions (Figure 1a).Our results showed that the SID oil contents displayed a gradual increase with maturity from 2.51% to 55.04% (Figure 1b).This was in line with a previous report that the oil content of mature SIDs ranges from 35 to 60% [3].The fluctuation may be correlated with climatic conditions, cultivation methods, extraction methods, and cultivars [2,16,17].Six major FA components of SID oil were palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and α-linolenic acid (C18:3) (Table 1).In early development, the saturated FAs of palmitic and stearic acid occupied more than half of the proportion (59.31%).As the seeds developed, PUFAs gradually became the main FA compositions, accounting for 78.92% at the termination stage of oil accumulation (Table 1).Interestingly, the percentage of PUFAs was lower than the previous report of 83.44%, while the percentage of oleic acid (10.98%) was higher than the previous report of 7.96% [13].It is undeniable that fluctuating climatic conditions inevitably lead to variations in the FA composition of oil seeds [16,17].Studies have reported that elevated PUFA levels in membrane lipids decrease the thermotolerance of plant cells [18].Thus, the lower PUFA content observed in this study might be due to the higher ambient temperatures in Hainan Island compared to Kunming [13], thereby enhancing plant thermotolerance.It is well established that oleic acid, a monounsaturated fatty acid (MUFA), is the principal precursor for PUFA biosynthesis [14,17].The inhibition of PUFA synthesis results in the accumulation of MUFAs.Note: Different letters represent significant differences, and shared letters represent no significant differences (p < 0.01).
It was clear that the highest content of C18:3 was in the sn-3 position, whereas the highest content of C18:2 was in the sn-2 position (Figure 2b).This result implied that the different positions of TAG might have different binding preferences for FA ligands.What was interesting about the data in Figure 2b was that, compared with Brassica napus [12] and Prunus sibirica [22], P. volubilis had higher C18:3 and C18:2 content in the sn-1 position of TAG.It seems that the sn-1 assembly of C18:3 and C18:2 may be important for high PUFA content in SID oil.Glycerol-3-phosphate is initially acylated by glycerol-3-phosphate acyltransferase (GPAT).Previous transcriptome profiling studies have identified five GPAT genes in SIDs [14].To develop oil crops with a high PUFA content, further investigation is required into the functional divergence of these GPAT members and their correlation with PUFA assembly at the sn-1 position.
Plants 2024, 13, x FOR PEER REVIEW 4 of 9 It was clear that the highest content of C18:3 was in the sn-3 position, whereas the highest content of C18:2 was in the sn-2 position (Figure 2b).This result implied that the different positions of TAG might have different binding preferences for FA ligands.What was interesting about the data in Figure 2b was that, compared with Brassica napus [12] and Prunus sibirica [22], P. volubilis had higher C18:3 and C18:2 content in the sn-1 position of TAG.It seems that the sn-1 assembly of C18:3 and C18:2 may be important for high PUFA content in SID oil.Glycerol-3-phosphate is initially acylated by glycerol-3phosphate acyltransferase (GPAT).Previous transcriptome profiling studies have identified five GPAT genes in SIDs [14].To develop oil crops with a high PUFA content, further investigation is required into the functional divergence of these GPAT members and their correlation with PUFA assembly at the sn-1 position.

Changes in the Ingredients of Phosphatidic Acid during Oil Accumulation
In the Kennedy pathway, the first two acylations of glycerol-3-phosphate produce phosphatidic acid (PA) [23].Then, the dephosphorylation of PA is catalyzed by phospatidate phosphatase to form DAG. A total of seven PA ingredients were identified in this study (Figure 3b and Supplementary Table S3).Interestingly, all the PA ingredients could match the DAG ingredients (Figure 3), which was inconsistent with the previous reports [12,22,24].The main difference here was that the SIDs had a high relative content of PUFAs in the PA ingredients, such as PA(18:3/18:3), PA(18:2/18:3), and PA(18:2/18:2) (Figure 3b).Nascent FAs from the plastid must be esterified to PC for further desaturation [25,26].The high content of these PA molecules in developing SIDs implied that those PUFAs desaturated on PC were released into the acyl-CoA pool, where they were  S2 and S3 for details.Values are represented as means ± standard deviations.Different letters above the groups represent significant differences, and shared letters represent no significant differences (p < 0.01).Abbreviations: diacylglycerol, DAG; phosphatidic acid, PA.

Changes in the Ingredients of Phosphatidic Acid during Oil Accumulation
In the Kennedy pathway, the first two acylations of glycerol-3-phosphate produce phosphatidic acid (PA) [23].Then, the dephosphorylation of PA is catalyzed by phospatidate phosphatase to form DAG. A total of seven PA ingredients were identified in this study (Figure 3b and Supplementary Table S3).Interestingly, all the PA ingredients could match the DAG ingredients (Figure 3), which was inconsistent with the previous reports [12,22,24].
The main difference here was that the SIDs had a high relative content of PUFAs in the PA ingredients, such as PA(18:3/18:3), PA(18:2/18:3), and PA(18:2/18:2) (Figure 3b).Nascent FAs from the plastid must be esterified to PC for further desaturation [25,26].The high content of these PA molecules in developing SIDs implied that those PUFAs desaturated on PC were released into the acyl-CoA pool, where they were assembled into the sn-1 and sn-2 positions of glycerol-3-phosphate through the Kennedy pathway.Thus, this pathway may play a prominent role in PUFA accumulation in developing SIDs.Also, the results of the PA ingredients further supported the idea of efficient assembly of PUFAs in the sn-1 position.
Plants 2024, 13, x FOR PEER REVIEW 5 of 9 pathway.Thus, this pathway may play a prominent role in PUFA accumulation in developing SIDs.Also, the results of the PA ingredients further supported the idea of efficient assembly of PUFAs in the sn-1 position.As expected, the 18:2/18:3 and 18:2/18:2 species had a high content both in DAG and PA (Figure 3).An interesting aspect was that the highest content of PA(18:3/18:3) was observed in mature SIDs, but the dephosphorylated production of DAG(18:3/18:3) only ranked sixth in DAG ingredients (Figure 3).A possible explanation for these results could be the substrate selection when using DAG for TAG biosynthesis [23].Combined with the highest content of TAG(18:3/18:3/18:3) (Figure 2a), we strongly believe that the SID cells may prefer to use DAG(18:3/18:3) as a substrate for the biosynthesis of TAG(18:3/18:3/18:3) during oil accumulation.Extensive use of DAG(18:3/18:3) led to its low ranking in DAG ingredients.Additionally, the highest content of DAG(18:2/18:3) was observed at 110 DAFs (Figure 3a), but the TAG(18:2/18:3/-) category did not occupy a seat in the main TAG ingredients (Figure 2a).This result suggested that the utilization rate of DAG(18:2/18:3) for TAG biosynthesis was low, supporting the conclusion of DAG substrate selection for TAG biosynthesis.

Plant Material
The P. volubilis specimens are preserved in the Museum of Beijing Forestry University, with the voucher number BJFC 00095003 (https://www.cvh.ac.cn/spms/detail.php?id= 9b9d48c8 (accessed on 1 April 2024)).After being introduced from Yunnan, the P. volubilis plants were cultivated in the plantation base of Hainan University, Danzhou, Hainan, China (latitude and longitude: 109.503179,19.542727).This species flowers and fruits year-round, allowing for the continuous collection of samples.Based on our previous investigation of SID development and oil accumulation [14], three crucial time points were selected for lipidome analysis.The three points represent the initiation (15 DAFs), rapid accumulation (70 DAFs), and culmination (110 DAFs) of seed oil accumulation.A short description is provided along with the BBCH code (Supplementary Table S5) [15].Fruits at stages 731, 787, and 799 were collected from the same tree.At least 25 fruit samples were collected for each period, and fresh seeds were obtained from star-shaped fruits.The samples were immediately flash-frozen in liquid nitrogen and stored at −80 • C until further analysis.

Oil Extraction and FA Analysis
Each sample was a composite formed by mixing together five seeds from the same stage.The mixed sample was freeze-dried using a Yamato DC401/800 freeze dryer (Chongqing, China) until a constant weight was achieved, and samples were then ground into a fine powder.The extraction of SID oil was conducted using Soxhlet extraction as previously reported [31].FA methyl esters were prepared and detected according to a previous experimental method [22].Heptadecanoic methyl ester (ANPEL, Shanghai, China) and GLC Mixture (ZZSRM, Shanghai, China) were used as the internal and external standards, respectively.The experiment was repeated three times.

Lipid Extraction
Using the same mixed sample as described above, lipid extraction was performed according to a previously reported method [32].Briefly, 480 µL MTBE:MeOH (5:1) and 200 µL ddH 2 O were sequentially added to 25 mg of SID samples in an EP tube.After a 30 sec vortex, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min in an ice-water bath.The homogenization and sonication cycles were repeated 3 times.Then, the samples were incubated at −40 • C for 1 h and centrifuged at 3000 rpm for 15 min at 4 • C. A total of 300 µL of supernatant was transferred to a fresh tube and dried in a vacuum concentrator at 37 • C.Then, the dried samples were reconstituted in 100 µL of 50% methanol in dichloromethane by sonication for 10 min in an ice-water bath.The constitution was then centrifuged at 13,000 rpm for 15 min at 4 • C, and 75 µL of supernatant was transferred to a fresh glass vial for liquid chromatography-tandem mass spectrometry (LC-MS/MS).The quality control sample was prepared by mixing an equal aliquot of the supernatants from all of the samples.
The QE mass spectrometer was used for its ability to acquire MS/MS spectra in a data-dependent acquisition (DDA) mode in the control of the acquisition software (Xcalibur 4.0.27,Thermo, Waltham, MA, USA).In this mode, the acquisition software continuously evaluates the full scan MS spectrum.The ESI source conditions were set as follows: sheath gas flow rate as 30 Arb, Aux gas flow rate as 10 Arb, capillary temperature as 320 • C (positive) or 300 • C (negative), full MS resolution as 70,000, MS/MS resolution as 17,500, collision energy as 15/30/45 in NCE mode, and spray voltage as 5 kV (positive) or −4.5 kV (negative), respectively.

Data Preprocessing and Statistical Analysis
The raw data files were converted to files in an mzXML format using the 'msconvert' program from ProteoWizard.Peak detection was first applied to the MS1 data.The CentWave algorithm in XCMS was used for peak detection with the MS/MS spectrum, and lipid identification was achieved through a spectral match using the LipidBlast library.
SPSS statistical software (version 19) was used for statistical analysis.Differences between groups were determined using a one-way analysis of variance (ANOVA) and multiplecomparison tests.A p value less than 0.01 was considered an extremely significant difference.

Figure 1 .
Figure 1.The developmental stages of P. volubilis fruits.(a) The fruits and seeds of P. volubilis at different developmental stages.The scale bar represents 1 cm.(b) The oil content of P. volubilis seeds during development.The oil was obtained by Soxhlet extraction.Values are represented as means ± standard deviations.Different letters above the groups represent significant differences, and shared letters represent no significant differences (p < 0.01).

Figure 1 .
Figure 1.The developmental stages of P. volubilis fruits.(a) The fruits and seeds of P. volubilis at different developmental stages.The scale bar represents 1 cm.(b) The oil content of P. volubilis seeds during development.The oil was obtained by Soxhlet extraction.Values are represented as means ± standard deviations.Different letters above the groups represent significant differences, and shared letters represent no significant differences (p < 0.01).

Figure 2 .
Figure 2. Relative content of the major TAG species during oil accumulation.(a) Relative contents of the major TAG species in SIDs at different developmental stages.Please refer to attached Table S1 for details.(b) Relative contents of C18:3 and C18:2 at different positions in mature SIDs.sn-1, sn-2, and sn-3, respectively, refer to different sites on the TAG.Values are represented as means ± standard deviations.Different letters above groups represent significant differences, and shared letters represent no significant differences (p < 0.01).

Figure 2 .
Figure 2. Relative content of the major TAG species during oil accumulation.(a) Relative contents of the major TAG species in SIDs at different developmental stages.Please refer to attached Table S1 for details.(b) Relative contents of C18:3 and C18:2 at different positions in mature SIDs.sn-1, sn-2, and sn-3, respectively, refer to different sites on the TAG.Values are represented as means ± standard deviations.Different letters above groups represent significant differences, and shared letters represent no significant differences (p < 0.01).

Figure 3 .
Figure 3. Relative contents of the major (a) DAG and (b) PA species found in SIDs during development.Please refer to attached TableS2and TableS3for details.Values are represented as means ± standard deviations.Different letters above the groups represent significant differences, and shared letters represent no significant differences (p < 0.01).Abbreviations: diacylglycerol, DAG; phosphatidic acid, PA.

Figure 3 .
Figure 3. Relative contents of the major (a) DAG and (b) PA species found in SIDs during development.Please refer to attached TablesS2 and S3for details.Values are represented as means ± standard deviations.Different letters above the groups represent significant differences, and shared letters represent no significant differences (p < 0.01).Abbreviations: diacylglycerol, DAG; phosphatidic acid, PA.

Figure 4 .
Figure 4. Relative contents of the major (a) PC and (b) LPC species found in SIDs during development.Please refer to attached TableS4for details.Values are represented as means ± standard deviations.Different letters above groups represent significant differences, and shared letters represent no significant differences (p < 0.01).Abbreviations: phosphatidyl choline, PC; lysophosphatidylcholine, LPC.

Figure 4 .
Figure 4. Relative contents of the major (a) PC and (b) LPC species found in SIDs during development.Please refer to attached TableS4for details.Values are represented as means ± standard deviations.Different letters above groups represent significant differences, and shared letters represent no significant differences (p < 0.01).Abbreviations: phosphatidyl choline, PC; lysophosphatidylcholine, LPC.

Table 1 .
Fatty acid components in developing SIDs.